凝血酶引致的血小板内钙,镁离子浓度及分布状态的变化

我们使用荧光探针ｆｕｒａ２、ｍａｇ－ｆｕｒａ２和ｆｌｕｏ３测定了凝血酶引致的血小板凝聚过程中细胞内钙、镁离子浓度的变化及分布状态。在０．５Ｕ／ｍｌ凝血酶作用下,血小板细胞内钙离子浓度呈双时相变化。血小板细胞群中细胞内钙离子浓度呈正态分布。伴随血小板凝聚时细胞内钙离子浓度增加,血小板细胞内游离镁离子浓度也明显增加,提示镁离子在血小板凝聚中有重要的作用。     

应用单分子荧光成像技术研究人参抗运动性疲劳分子机制

采用单分子荧光成像技术,通过建立运动性疲劳的细胞模型对中药人参抗运动性疲劳的分子机制进行研究。取新生大鼠骨骼肌细胞,将其随机分成人参组和对照组。在两组细胞的培养基中分别加入2mg／mL人参提取物和等量的D—hanks液进行培养。两组细胞分化成肌管后,将用钙离子染料处理过的细胞置于共聚焦显微镜上检测其荧光强度。待荧光强度稳定时,分别向两组细胞加1μmol／L地塞米松溶液,记录并比较两组细胞用地塞米松刺激前后荧光强度的改变幅度。结果发现,两组细胞胞浆内钙离子的荧光强度均明显增强（P〈0．05）,但人参提取物组细胞胞浆内钙离子浓度的增加幅度明显低于对照组（P〈0．05）。由此可见,人参提取物对地塞米松刺激引起的疲劳骨骼肌细胞具有保护作用,人参消除运动性疲劳的机制可能与其调节骨骼肌细胞胞浆内钙离子浓度有关。     

54kD钙结合蛋白对血小板膜磷脂酶A2的影响

用钙离子沉淀和柱层析的方法从猪血小板中分离纯化到了一个分子量为５４ｋＤ、具有Ａｎｎｅｘｉｎ样生物学作用的钙结合蛋白,并用对钙离子有高度特异亲和力的偶氮胂试剂确定了该蛋白质具有钙结合能力。为了研究５４ｋＤ钙结合蛋白对猪血小板膜磷脂酶Ａ２的作用,我们用密度梯度离心方法制备了猪血小板膜,并用其作为酶和底物的材料,用游离脂肪酸的微量显色法建立了测定猪血小板膜结合的ＰＬＡ２活性的方法。我们的实验结果表明：５４ｋＤ钙结合蛋白在反应体系钙离子浓度低于１ｍｍｏｌ／Ｌ时,对膜结合的ＰＬＡ２具有激活作用;在钙离子大于１ｍｍｏｌ／Ｌ时;对膜结合的ＰＬＡ２具有抑制作用。并且抑制作用不随底物浓度的升高而降低．     

低强度He-Ne激光对红细胞胞浆内游离钙离子浓度的影响

低强度He-Ne激光对红细胞变形性的影响已得到广泛的认可和应用,但其具体调节机制尚不明确,通过研究低强度He-Ne激光对红细胞胞浆内钙离子浓度的影响,探讨其对红细胞变形性影响的机制。采用A23187处理过的红细胞,分别用5 mW和9 mW激光照射后,观察红细胞胞浆内钙离子浓度变化。结果显示:低强度He-Ne激光照射后的红细胞与无照射组的红细胞相比,红细胞胞浆内钙离子浓度显著降低,但在本实验中钙离子浓度的降低与激光照射剂量无显著相关。由此得出结论:降低红细胞胞浆内钙离子浓度可能是低强度He-Ne激光调节红细胞变形性的重要机制。     

荧光探针测定人血小板内游离钙浓度及抗钙药物的影响

Quin 2是一种对钙离子敏感的荧光素。它的乙酰化形式Quin2-AM具有亲脂性,可穿过细胞膜进入细胞内,水解后可特异性地与细胞内钙离子结合发出荧光。我们利用Quin 2对人血小板静息状态下及加凝血酶后激动状态下的血小板内钙离子浓度进行了     

白细胞介素-1β对骨髓基质细胞膜电位及细胞内Ca2+和pH的影响

采用荧光染料 Ｄｉ Ｂ Ａ Ｃ４（３）, Ｆｌｕｏ３／ Ａ Ｍ 和 Ｓ Ｎ Ａ Ｌ Ｆ Ｃａｌｃｅｉｎ／ Ａ Ｍ 分别标记小鼠骨髓基质细胞（ Ｂ Ｍ Ｓ Ｃ）,在激光扫描共聚焦显微镜下直接监测重组人白细胞介素１β（ Ｉ Ｌ１β）刺激后细胞膜电位,细胞内游离 Ｃａ２＋ 浓度和胞浆 ｐ Ｈ 的实时动态变化． 结果发现： Ｉ Ｌ１β加入测定体系后浓度依赖性地引起 Ｂ Ｍ Ｓ Ｃ 膜电位的迅速改变． 低浓度时发生去极化反应,高浓度时发生超极化反应． 非受体方式作用的 Ｉ Ｌ１β肽段 １６３１７１ 对膜电位无影响． Ｉ Ｌ１β不影响细胞内 Ｃａ２＋ 的浓度和胞浆ｐ Ｈ． 研究表明膜电位的变化为 Ｉ Ｌ１ 受体后早期事件,它与细胞内 Ｃａ２＋的浓度和胞浆 ｐ Ｈ 的调节无关．     

凝血酶对荧光标记的人单个血小板游离[Ca^2＋]和膜电位的动态影响

本文将ｆｌｕｏ－３和ｄ_ｉ－ＢＡ－Ｃ４（３）荧光标记的血小板固定于纤维蛋白原表面,以５７０型粘附式细胞仪（ＡＣＡＳ５７０）动态观察了凝血酶激活的人单个血小板细胞内游离［Ｃａ~（２＋）］（钙离子浓度）和膜电位的变化。静息状态时细胞游离［Ｃａ~（２＋）］和膜电位荧光较低,波动不明显。当加入０．１Ｕ／ｍｌ凝血酶激活时,［Ｃａ~（２＋）］（细胞内游离钙离子浓度）与膜电位迅速升高,随后［Ｃａ~（２＋）］出现反复振荡,幅度达约５００荧光单位,而膜电位基本上保持峰值水平。［Ｃａ~（２＋）］_ｉ升高与膜电位变化在时间和程度上不一致。本文结果提示,凝血酶引起血小板［Ｃａ~（２＋）］振荡和膜去极化,后者不是Ｃａ~（２＋）内流引起的。     

激光扫描共聚焦显微镜术检测单个血小板钙波动方法的探讨

目的 探讨血小板贴壁的简易方法,同时,观察单个血小板激活前后细胞内钙波动及形态变化的规律。方法 采用多种粘附剂固定血小板,利用钙荧光探针（Fluo-3/AM)（终浓度10－20μmol)进行染色,在激光扫描共聚焦显同镜检测下加入二磷酸腺苷,观察和分析血小板内Ca^2 浓度及形态变化。结果 多聚赖氨酸促进血小板贴壁固定的效果最佳;单个血小板活后细胞内Ca^2 浓度为激活前的128％,形态圆形或椭形转为不规则形,有空泡,突起形成。结论 多聚赖氨酸是血小板贴壁的理想粘附剂,本方法能简易,快捷地监测血小板激活过程中胞浆内钙离子动态变化及形态改变,有助于血小板功能的深入研究。     

血小板激活时胞质游离钙浓度与两种颗粒数变化的相关性

用电镜形态计量法检测血小板α颗粒（αＧ）和致密颗粒（ｄＧ）的数密度,用钙荧光指示剂Ｆｕｒａ２检测血小板胞质游离Ｃａ＾２＋浓度（「Ｃａ＾２＋」ｉ）,观察到在钙离子导体Ａ２３１８７作用下,血小板「Ｃａ＾２＋」ｉ明显升高。凝血酶与ＡＤＰ也都分别引起「Ｃａ＾２＋」ｉ升高,且有浓度依赖性,选用三种激动剂的不同量以反映血小板不同程度激活时,测定「Ｃａ＾２＋」与颗粒数密度,分析两者间的相关性,发现αＧ和ｄＧ的数     

构建钙荧光探针细胞用于探究胞浆和线粒体钙对ATP刺激的响应

目的:构建表达基因编辑钙探针(GECIs)的细胞系HeLa-GECIs,探究细胞应答外界ATP刺激中钙离子在细胞内的响应和变化。方法:分别用能够直接通过荧光强度反映细胞胞浆内和线粒体内钙离子相对浓度的2种钙探针cyto-GCaMP6和4mt-GCaMP6感染HeLa细胞,获得2种表达钙离子探针的HeLa细胞系;在感染了2种腺病毒探针24 h后,用共聚焦荧光显微镜检测荧光探针在HeLa细胞内的表达情况;在表达2种钙探针的细胞的培养基中加入外源ATP,用Time-lapse成像动态观测技术观察HeLa细胞内钙离子对外环境中ATP的响应。结果:共聚焦荧光显微镜观察,确定95%以上的细胞表达了对应的钙离子指示荧光探针;Time-lapse成像动态观测技术观察发现,在细胞培养基中加入ATP后,细胞胞浆钙探针荧光强度瞬时(3～6 s)升至10倍,200 s后逐渐降低到基础水平;线粒体钙到达峰值(4倍)的时间稍滞后(5～8 s),并且回落更慢,300 s时至1.5倍。在ATP受体P2X7抑制剂A438079预处理的实验组,上述胞浆钙和线粒体钙浓度上升不明显。结论:构建了能在活体细胞内通过荧光探针实时监测钙离子响应胞外ATP刺激的细胞实验体系,为进一步深入探究ATP等危险信号导致细胞的炎性损伤机制奠定了基础。     

The role of Ca2+ in regulating the catabolism of PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in rabbit platelets.

In the present study we have investigated the effect of changes in the concentration of cytosolic free Ca2+ ([Ca2+]i) on the deacetylation-reacylation of PAF-acether (alkylacetylglycerophosphocholine, alkylacetyl-GPC) by rabbit platelets. Washed platelets were incubated with alkyl[3H]acetyl-GPC ([3H]acetyl-PAF) or [3H]alkylacetyl-GPC ([3H]alkyl-PAF) and [Ca2+]i was subsequently elevated by the addition of the ionophore A23187 or thrombin. The catabolism of PAF-acether was studied by measuring the release of [3H]acetate or the formation of [3H]alkylacyl-GPC. The ionophore inhibited the release of [3H]acetate and the formation of [3H]alkylacyl-GPC with no accumulation of lyso-[3H]PAF, indicating that the deacetylation of PAF-acether was blocked. The effect of ionophore on the deacetylation of PAF-acether was parallel with the increase of [Ca2+]i and could be reversed by the addition of EGTA. In contrast with the prolonged inhibition evoked by ionophore, thrombin, which induced a transient elevation of [Ca2+]i, merely delayed the deacetylation of PAF-acether. Since intact platelets failed to convert exogenous lyso-PAF, the effect of Ca2+ on its acylation was investigated by using platelet homogenates. These experiments showed that the acylation of lyso-PAF was inhibited by the exogenously added Ca2+, with a maximum effect at 1 mM. When the formation of endogenous lyso-PAF from the labelled pool of alkylacyl-GPC was examined, a prolonged increase in the concentration of lyso-PAF with a parallel and equally prolonged decrease in the cellular level of alkylacyl-GPC were observed after the addition of ionophore to intact platelets. The addition of EGTA reversed the effect of ionophore, thus permitting reacylation of lyso-PAF. In contrast, only a transient change in the level of lyso-PAF and alkylacyl-GPC was evoked by the addition of thrombin. Therefore we conclude that the inhibitory effect of Ca2+ on the deacetylation-reacylation of PAF-acether may have an important role in the regulation of its biosynthesis.     

Lack of inhibition of thrombin-induced rise in intracellular Ca2+ levels and 5-hydroxytryptamine secretion by 1-oleoyl-2-acetylglycerol in human platelets

The effect of 1-oleoyl-2-acetylglycerol (OAG) on the thrombin-induced rise in intracellular Ca2+ levels ([Ca2+]i) and 5-hydroxy[14C]tryptamine ([14C]5HT) secretion was studied. In washed human platelets prelabelled with [14C]5HT and quin 2, OAG (10-50 micrograms/ml) induced no significant aggregation, [14C]5HT secretion or rise in [Ca2+]i in the presence or absence of fibrinogen. However, addition of OAG (10-50 micrograms/ml) 10 s to 5 min before or 10-60 s after addition of threshold concentrations of thrombin (less than 0.03 U/ml) resulted in a significant potentiation of aggregation and [14C]5HT secretion without any effect on the thrombin-induced rise in [Ca2+]i. Both EGTA, which abolished the latter and creatine phosphate/creatine phosphokinase, the ADP scavenger, totally inhibited the aggregation but only partially reduced [14C]5HT secretion in response to thrombin plus OAG. At higher concentrations of thrombin, neither the rise in [Ca2+]i nor the extent of [14C]5HT secretion was significantly altered by OAG addition. The results demonstrate that, unlike phorbol esters, OAG has no inhibitory effect on thrombin-induced [Ca2+]i mobilisation but can synergize with low concentrations of thrombin in potentiating [14C]5HT secretion even at basal [Ca2+]i.     

Effects of guanine nucleotides on the properties of 5-hydroxytryptamine secretion from electropermeabilised human platelets

Guanosine 5'-[gamma-thio]triphosphate and guanosine 5'-[beta,gamma-imido]triphosphate enhance Ca2+-dependent 5-hydroxytryptamine secretion from electropermeabilised human platelets. GTP has little such effect except when the platelets are permeabilised, and incubated with this nucleotide, at 2 degrees C and pH 7.4. The lag phase observed in the time course of 5-hydroxytryptamine secretion induced by addition of guanosine 5'-[gamma-thio]triphosphate is markedly longer than that characterising secretion induced by Ca2+ alone, by thrombin +/- GTP or by guanosine 5'-[gamma-thio]triphosphate in the presence of thrombin. GTP causes competitive inhibition of the enhancement of the Ca2+-dependent secretory response induced by guanosine 5'-[gamma-thio]triphosphate when both nucleotides are added simultaneously. The extent of inhibition is decreased if guanosine 5'-[gamma-thio]triphosphate is added prior to GTP. GTP markedly enhances the effect of thrombin on Ca2+-dependent 5-hydroxytryptamine secretion by increasing the maximal extent of the response and decreasing the thrombin concentration required to give half-maximal response. A similar effect is observed on addition of guanosine 5'-[gamma-thio]triphosphate in the presence of thrombin at short incubation times. On more prolonged incubation the effects of thrombin and guanosine 5'-[gamma-thio]triphosphate are additive. Guanosine 5'-[beta-thio]diphosphate completely inhibits the response induced by guanosine 5'-[gamma-thio]triphosphate or guanosine 5'-[beta,gamma-imido]triphosphate but has little effect on the response induced by Ca2+ when added alone or in the presence of thrombin. Partial inhibition is observed for the response induced by thrombin + GTP. Cyclic-AMP effectively inhibits the response induced by thrombin + GTP but has little effect on that induced by guanosine 5'-[gamma-thio]triphosphate or guanosine 5'-[beta,gamma]imidotriphosphate. The results provide further support for the proposal [Haslam, R.J. & Davidson, M.M.L. (1984) FEBS Lett. 170, 90-95], that receptor--phospholipase-C coupling in platelets is mediated in part by a guanine-nucleotide-binding (Np) protein but that a coupling mechanism may also exist which is independent of such a protein. The properties of guanine-nucleotide-dependent coupling resemble those previously described for receptor--adenylate-cyclase coupling.     

槲皮素硫酸酯对猪血小板胞浆游离钙浓度和蛋白激酶C的影响

槲皮素（ｑｕｅｒｃｅｔｉｎ,Ｑｕｅ）,是一种天然的黄酮类化合物,具有多种生物活性［１］,但是Ｑｕｅ水溶性差,口服时胃肠难以吸收［２］．因此,为进一步开发和利用Ｑｕｅ,人工合成水溶性Ｑｕｅ——槲桷皮素硫酸酯（ｓｏｄｉｕｍｑｕｅｒｃｅｔｉｎｓｕｌｆａｔｅ...     

Two thrombin-activated Ca2+ channels in human platelets.

The regulation of extracellular Ca2+ entry into fura-2-loaded human platelets was examined following stimulation with thrombin. In the presence of external Ca2+, stimulation of platelets with thrombin resulted in a rapid increase, followed by a plateau, in intracellular Ca2+ concentration ([Ca2+]i). Pretreatment with wortmannin, a specific inhibitor of myosin light chain kinase, suppressed only the plateau phase and had no effect on the initial rapid increase in [Ca2+]i. In Ca(2+)-free EGTA buffer, thrombin induced a transient and relatively small increase in [Ca2+]i caused by Ca2+ release from internal stores. When Ca2+ was added subsequently to the Ca(2+)-free medium within 10 min after thrombin activation, a marked increase in [Ca2+]i was seen, reflecting thrombin-stimulated external Ca2+ entry. With the Ca(2+)-free medium, wortmannin did not affect either the Ca2+ mobilization from the internal stores or the rapid external Ca2+ entry at early time points (within 5 s) after thrombin stimulation, whereas it significantly inhibited Ca2+ entry when Ca2+ was added later (at 3 min). Wortmannin inhibition of this late Ca2+ entry and that of 20-kDa myosin light chain phosphorylation after thrombin stimulation were dose- and preincubation time-dependent and correlated well with each other. These results suggest that two different channels are responsible for Ca2+ entry in human platelets at the early and late phases of thrombin stimulation and that the channel responsible for the late phase of Ca2+ entry may be activated by a mechanism involving myosin light chain kinase.     

Lack of evidence for voltage dependent calcium channels on platelets

Intracellular calcium was measured in human platelets using the fluorescent calcium indicator Quin 2. A concentration dependent increase was observed with thrombin. Depolarisation induced by high KCl concentrations did not alter [Ca++]i. The calcium agonist Bay K 8644 did not affect resting levels or thrombin stimulated elevation of intracellular calcium. The calcium antagonists diltiazem, verapamil and PN 200-110 did not inhibit the thrombin stimulated elevation in [Ca++]i. Pretreatment of platelets with adenylate cyclase stimulants reduced the rate and magnitude of the maximal [Ca++]i elevation due to thrombin. In addition, thrombin stimulation of 45Ca++ influx was insensitive to Bay K 8644, verapamil, diltiazem and Pn 200-110. We conclude that functional voltage sensitive calcium channels are not present on human platelets.     

Secondary signals mediated by GPIIb/IIIa in thrombin-activated platelets

We have previously found that stimulation of aequorin-loaded platelets by thrombin produced a two-peaked increase in intracellular free calcium concentration ([Ca2+]i), and the development of the second peak of [Ca2+]i was closely related with the aggregation. In this report, we studied the interrelationship between the GPIIb/IIIa complex, aggregation, cytoskeletons and [Ca2+]i of platelets. The pretreatment of the platelets with dihydrocytochalasin B (4 microM), an actin polymerization inhibitor, did not inhibit aggregation and TXB2 production, but did inhibit both actin polymerization and the second peak of [Ca2+]i increase induced by thrombin, suggesting that actin polymerization and the second peak of [Ca2+]i are interrelated. GRGDSP (100 microM), a synthetic anti-adhesive peptide, has already been reported to inhibit platelet aggregation and the second peak of [Ca2+]i induced by thrombin. It also inhibited actin polymerization and TXB2 production, suggesting that aggregation was important for not only the generation of the second peak of [Ca2+]i but also for actin polymerization and TXB2 production. PGI2 (5 nM) did not abolish but only delayed aggregation, TXB2 production, actin polymerization and the second peak of [Ca2+]i increase. These findings suggest that the secondary signals are caused by aggregation (fibrinogen-binding to the GPIIb/IIIa) in thrombin-aggregated platelets, which results in the TXA2 production and the secondary peak of [Ca2+]i increase, and the latter was dependent on actin polymerization.     

Thrombin and ionomycin can raise platelet cytosolic Ca2+ to micromolar levels by discharge of internal Ca2+ stores: studies using fura-2

In the presence of 1 mM EGTA, the addition of the calcium ionophore ionomycin to human platelets loaded with 30 microM fura-2 could elevate [Ca2+]i from less than 100 nM to a maximum of greater than 3 microM, presumably by discharge of Ca2+ from internal stores. Under the same conditions thrombin could maximally increase [Ca2+]i to a peak of greater than 1 microM which then declined to near resting levels within 3-4 minutes; by contrast in platelets loaded with 1 mM quin2 thrombin could raise [Ca2+]i to only about 200 nM. In the presence of 1 mM Ca2+ the peak response to thrombin in fura-2-loaded platelets was higher (1.4 microM) than that observed in the presence of EGTA (1.1 microM) and the elevation in [Ca2+] was prolonged, presumably by Ca2+ influx. These results with fura-2-loaded platelets indicate that mobilisation of internal Ca2+ can contribute a substantial proportion of the early peak [Ca2+]i evoked by thrombin directly confirming the deductions from previous work with different loadings of quin2. Under natural conditions the major role of Ca2+ influx may be to prolong the [Ca2+]i rise rather than to make it larger.     

Fluorescence digital image analysis of thrombin and ADP induced rise in intracellular Ca2+ concentration of single blood platelets.

Cytoplasmic free Ca2+ concentration, [Ca2+]i, was estimated in single rabbit blood platelets by digital imaging microscopy with the use of the specific Ca(2+)-indicator dye Fura-2. Uneven distribution and low level of [Ca2+]i was found in the resting platelet even in the presence of extracellular 1 mM Ca2+. Thrombin at 1 unit/ml immediately caused a transient increase in [Ca2+]i, which was followed by a secondary and sustained increase in [Ca2+]i. The distribution of increased levels of [Ca2+]i was also shown to be uneven within the cell. The presence of 1 mM EGTA in the medium only slightly decreased the initial rise in [Ca2+]i, but completely inhibited the latter phase, a sustained rise in [Ca2+]i. This result shows that the initial rise of [Ca2+]i might not be caused by Ca2+ influx, but might be induced by mobilization of Ca2+ from intracellular Ca2+ storage sites. This speculation is further supported by the fact that the elevated [Ca2+]i induced by thrombin immediately decreased to the base line value when 3 mM EGTA was applied. Thus, thrombin induced elevation of [Ca2+]i is suggested to consist of two different processes, namely the mobilization of Ca2+ from the intracellular storage sites and the successive Ca2+ influx through the receptor activated Ca2+ channels. Stimulation with ADP also caused a rapid elevation of platelet [Ca2+]i, but this effect of ADP was different form that of thrombin. Thus, the ADP induced rise in [Ca2+]i was accompanied by oscillation and was inhibited by extracellular EGTA. Our present experiment is the first report that clearly and directly reveals the differences between the effects of thrombin and ADP on [Ca2+]i of platelets.     

Synergistic potentiation of 5-hydroxytryptamine secretion by platelet agonists and phorbol myristate acetate despite inhibition of agonist-induced arachidonate/thromboxane and beta-thromboglobulin release and Ca2+ mobilization by phorbol myristate acetate.

Previous studies have demonstrated an inhibition of agonist-induced inositol phospholipid breakdown and intracellular Ca2+ ([Ca2+]i) mobilization by phorbol esters in platelets. In this study, we have examined the effect of phorbol 12-myristate 13-acetate (PMA) on agonist-induced granule secretion and correlated it with agonist-induced [Ca2+]i mobilization, arachidonate and thromboxane (Tx) release in human platelets. With increasing times of incubation with PMA (10 s-5 min), the rise in [Ca2+]i induced by thrombin and the TxA2 mimetic, U46619, was increasingly inhibited (90-100% with 5 min incubation) and, correlating with this, thrombin-induced [3H]arachidonate, TxB2 and beta-thromboglobulin (beta TG) release were also inhibited. In addition, the conversion of exogenously added arachidonate to TxB2 was inhibited (50-80%) by a 10 s-5 min pretreatment with PMA. However, secretion of 5-hydroxy[14C]tryptamine (5HT) induced by thrombin or U46619 was not inhibited by 10 s-2 min incubations with PMA and, on the contrary, with low agonist concentrations, was potentiated by PMA in the absence of a significant rise in [Ca2+]i or endogenous Tx formation, to levels significantly greater than or equal to the sum of that obtained when agonist and PMA were added separately. With longer times of incubation with PMA (5 min), these synergistic effects became less pronounced as inhibitory effects of PMA on agonist-induced [14C]5HT secretion became apparent. The results indicate that, while PMA may cause an inhibition of agonist-induced [Ca2+]i mobilization resulting in an inhibition of agonist-induced arachidonate, TxB2 and beta TG release, its effects on agonist-induced 5HT secretion may be complicated by [Ca2+]i-independent synergistic effects of agonist and PMA.